Objectives: To test the effect of Paclitaxel and Bevacizumab on the tumor blood vessels as well as their effects on the microdistribution of Alexa Fluor 647-B3 in tumors by fluorescence microscopy. We previously found that a combination therapy of Y-90-B3 with Paclitaxel produced a synergistic effect in the shrinkage of tumor size whereas the addition of Avastin treatment to the combined therapy of 60 microCi B3 and Taxol provided some additive effect in survival. While the control mice had a median survival time of 5 days, the Taxol and Avastin treatment delayed tumor growth with a median survival time of 17 days and 11 days, respectively. The Y-90 B3 treatment showed a dose-dependent response with a median survival time of 18 days for the 60 microCi B3 group and 29 days for the 100 microCi B3 group. The addition of Avastin before 60 microCi B3 treatment produced an additive effect in survival with a median survival time of 20 days. The combined therapy involving 60 microCi B3 and 100 microCi B3 with Taxol showed a striking synergistic effect in shrinking tumor and prolonging the survival time. On day 120, 3 of 9 mice (33%) treated with a combined therapy involving 60 microCi B3 and Taxol, and 6 of 6 mice treated with a combined therapy of 100 microCi B3 with Taxol were alive with no tumor. The addition of Avastin treatment to the combined therapy of 60 microCi B3 and Taxol provided some additive effect in survival; 3 of 6 (50%) treated with Avastin were alive with no tumor. Based on these previous studies, we investigated the effect of Paclitaxel and Bevacizumab on the tumor blood vessels as well as their effects on the microdistribution of Alexa Fluor 647-B3 in tumors by fluorescence microscopy. Methods: Groups of nude mice (n = 45 mice/group) were inoculated s.c. with A431 tumor cells expressing the Le-Y antigen on the right hind flank. When the tumor size reached 200 cubic mm, the tumor-bearing mice were divided into three groups and were injected with 1) i.v. Alexa Fluor 647-conjugated B3 (150 micro-g in 0.2 ml of PBS) alone on day 0, 2) i.v. Alexa Fluor 647-B3 on day 0 followed by i.p. Paclitaxel (40 mg/kg in 0.2 ml of normal saline) on day 1, and 3) i.v. Bevacizumab (5 mg/kg in 0.2 ml of PBS) on day 0 followed by i.v. Alexa Fluor 647-B3 on day 1 to investigate the effect of Paclitaxel and Bevacizumab on the tumor microdisribution of Alexa Fluor 647-B3. Two days after the injection of Alexa Fluor 647-B3, the mice received a lateral tail vein injection of rhodamine-lectin (RCA, 1 mg in 0.2 ml of PBS) to delineate the blood vessels and 5 min after the lectin injection, the mice were euthanized by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Tumors were harvested with intact skin and flash-frozen using liquid nitrogen for subsequent sectioning and staining. Tumors were sectioned using a Leica CM1850 cryostat at 8 micro-m thickness in 3 different regions to obtain representative sections throughout the tumor. Tumor sections were fixed with formalin for 20 min and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). Imaging was performed with a 10X objective (pixel size = 0.64 micro-m, binning 2x2) using an epi-fluorescent microscope (Zeiss, Axio Imager.M1, Thornwood, NY) equipped with a motorized scanning stage and mosaic stitching software (Axiovision, Zeiss). Three independent channels were obtained: DAPI for nuclei (shown in blue), Rhodamine for blood vessels (shown in red), and Cy5 for Alexa Fluor 647-B3 antibody (constant exposure time of 40 ms, shown in green). A tumor that did not contain B3 antibody was imaged with identical parameters to obtain background signal intensity. Image analysis was performed with a custom-designed MATLAB script (MathWorks, Natick, MA). Individual image channels were exported from Axiovision as 16-bit grayscale tiff images to Photoshop where a tumor region was isolated to create a tumor mask. The tumor mask, blood vessel image and B3 antibody image were loaded into MATLAB and a tumor blood vessel mask and distance map were created. Overall B3 antibody intensity and penetration from the tumor edge and blood vessel surface were calculated with a background intensity subtraction. In addition, vascular parameters and architecture such as micro-vasculature density (MVD), blood vessel size, and median distance from a tumor pixel to the nearest vascular surface were measured. Values were grouped together from the 3 tumor regions to represent a tumor. Each tumor was treated as an independent sample (n= 4-5, one tumor was removed due to excessive necrosis limiting analysis). Results: The fluorescence microscopic determination revealed that Paclitaxel (40 mg/kg) treatment did not significantly change the MVD (67.4 vs 70.3 vessels/mm-squared for the control, P > 0.05), median blood vessel area (92.7 vs 100.1 micro-m-squared for the control), and the median distance to the nearest vascular surface, an indicator of vascular architecture (78.8 vs 69.3 micro-m for the control, P > 0.05) compared to the control one day after the treatment. Comparatively, the treatment with Bevacizumab decreased the MVD (53.4 vessels/mm- squared) by 24% and increased the median distance (95.1 micro-m) from a tumor pixel to the nearest vascular surface by 37% compared to the control, although it is not statistically significant (P > 0.05). Bevacizumab did not change the median blood vessel size (92.22 micro-m-squared). Antibody accumulation was determined by analyzing fluorescence intensity in tumor sections. The analysis qualitatively suggests that B3 antibody accumulation and penetration were improved with Paclitaxel treatment and reduced with Bevacizumab treatment. The image analysis demonstrated that when 150 micro-g of fluorescence-labeled B3 was administered, the highest concentration of fluorescence labeled B3 (2 fold higher than that in the tumor surface) accumulated at 50 micro-m distance from the tumor surface. The B3 concentration decreased rapidly as it moved toward the tumor center: The B3 concentration at 1 mm from the tumor surface became 4 fold lower than the maximum concentration at 50 micro-m from the tumor surface. The fluorescence microscopy study also showed that the B3 concentration diminished rapidly as moving away from blood vessels, and a steady state concentration of mAb was shown at a distance between 50 to 100 micro-m from blood vessels. This steady state mAb concentration was approximately 3.4 fold less than the concentration of mAb in tumor blood vessel surface. The Paclitaxel treatment significantly improved B3 antibody delivery to the tumor by 45% (2570 vs 1775 arbitrary fluorescence units for the control, P < 0.05). The B3 penetration was significantly improved following treatment with Paclitaxel both from the tumor surface and from the blood vessel surface (P < 0.05). The Paclitaxel treatment increased the steady state concentration of B3 by 60% (P < 0.05) compared to that of the control. The Paclitaxel treatment also appeared to impact the integrity of the tumor tissue with a muddled cell nuclei appearance. Comparatively, Bevacizumab had a profound effect on B3 antibody delivery, significantly reducing the accumulation by 96% (73 vs 1775 for the control, P < 0.05). In addition, Bevacizumab significantly reduced the penetration of B3 antibody (P < 0.05). Conclusion: The positive effect of Paclitaxel on the accumulation and penetration of B3 warrants further studies on the effect of the dose of B3 and Paclitaxel on the accumulation and penetration of B3 in larger tumors. The influence of tumor microdistribution on combined tumor radioimmunotherapy regimens represents a promising area for further investigation and optimization to improve radioimmuno-therapy for solid tumors in the clinic.